Influence of Galactomannans with Different Molecular Weights on

Biomacromolecules 2005, 6, 3291-3299

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Influence of Galactomannans with Different Molecular Weights on the Gelation of Whey Proteins at Neutral pH So´ nia R. Monteiro,† Cla´ udia Tavares,† Dmitry V. Evtuguin,‡ Nuno Moreno,§ and J. A. Lopes da Silva*,† Departamento de Quı´mica, Universidade de Aveiro, 3810-193 Aveiro, Portugal, CICECO, Universidade de Aveiro, 3810-193 Aveiro, Portugal, and Cell Imaging Unit, Instituto Gulbenkian de Cieˆ ncia, 2780-156 Oeiras, Portugal Received May 13, 2005; Revised Manuscript Received July 12, 2005

The effect of locust bean gum, a galactomannan, with different molecular weights on the microstructure and viscoelastic properties of heat-induced whey protein gels has been studied using confocal laser scanning microscopy and small-deformation rheology. The results obtained clearly showed that differences in the molecular weight of the polysaccharide have a significant influence on the gel microstructure. Homogeneous mixtures and phase-separated systems, with dispersed droplet and bicontinuous morphologies, were observed by changing the polysaccharide/protein ratio and/or the molecular weight. At 11% whey protein, below the gelation threshold of the protein alone, the presence of the nongelling polysaccharide induces gelation to occur. At higher protein concentration, the main effect of the polysaccharide was a re-enforcement of the gel. However, at the higher molecular weight and concentration of the nongelling polymer, the protein network starts to lose elastic perfection, probably due to the formation of bicontinuous structures with lower connectivity. 1. Introduction Whey proteins (WP) have widespread uses as food ingredients due to their unique nutritional and functional properties.1 The heat-induced gelation properties of these complex protein systems have been studied extensively.2-7 Gelation is usually achieved upon heating above the protein’s denaturation temperature; aggregates and/or gels result from a two-step sequential process including partial denaturation and unfolding of the proteins and aggregation reactions.8 Different network structures are obtained by changing protein concentration, pH, ionic strength, solutes, and gelation kinetics. At a pH close to the isoelectric point (pI) of the proteins, the aggregation reactions start prior to denaturation, resulting in the so-called particulate gels characterized by opaque systems with protein-rich domains with a diameter of the order of micrometers.9 In the present study, the WP gelation was studied at neutral pH (i.e., above the pI) and low ionic strength. Under these conditions, the proteins are highly charged, and more extensive protein unfolding and much stronger hydrogen bonds between polypeptide chains occur,10 leading to the formation of transparent fine-stranded gels.2 In recent years, considerable interest has been devoted to the study of polysaccharide-protein mixtures,11-13 particularly as a way to understand multicomponent complex food systems and to optimize new food ingredients. Polymer * Corresponding author. Phone: +351-234370360. Fax: +351234370084. E-mail: [email protected]. † Departamento de Quı´mica, Universidade de Aveiro. ‡CICECO, Universidade de Aveiro. §Cell Imaging Unit, Instituto Gulbenkian de Cie ˆ ncia.

incompatibility is widely accepted as a general phenomenon in aqueous protein-polysaccharide systems,11,14-16 especially with high molecular weight biopolymers and in the absence of complex coacervation. This well-illustrated thermodynamic incompatibility arises mainly from the low entropy of mixing and results in segregation and phase separation, with both macromolecules tending to demix into proteinrich and polysaccharide-rich regions. The occurrence and extension of demixing is influenced by a wide range of intrinsic and extrinsic factors, including biopolymer concentration, molecular weight, conformation, charge density, and therefore, pH and ionic strength of the solvent,17 and it is expected to greatly affect the rheological behavior of the multicomponent system. Several studies have been made on the gel formation of whey proteins in combination with polysaccharides, focusing mainly on the effect of ionic gelling18-22 or nongelling polysaccharides23-26 and on the effect of starch and starchderived gelling polysaccharides.27-29 In comparison, fewer studies have been reported on the influence of neutral nongelling polysaccharides on whey protein gelation.14,30-33 The presence of galactomannan chains was shown to influence the formation and viscoelastic behavior of both particulate (in the vicinity of the protein’s isoelectric point) and fine-stranded (around neutral pH) whey protein gels.32,33 However, the influence of the molecular characteristics of the polysaccharide on the gelling behavior of protein systems has been an almost unexplored subject. In this work, we have studied the influence of the molecular mass of a nongelling neutral polysaccharide on whey protein gelation, focusing mainly on the heat-induced

10.1021/bm050331+ CCC: $30.25 © 2005 American Chemical Society Published on Web 08/30/2005

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gelation of the mixtures at pH 7, gel curing, and viscoelasticity and microstructure of the cured gels. Two different protein concentrations have been analyzed, one close to the sol-gel transition threshold (11% protein) and the other corresponding to a more elastic and well-developed gel (14% protein). The polysaccharide samples were galactomannans from locust been gum, native and depolymerized by enzymatic treatment with an endo-β-mannanase, at concentrations between 0 and 0.7% (w/w). 2. Experimental Section 2.1. Materials. Whey protein isolate (WPI) was an undenatured and virtually lactose-free preparation, (BiPRO, 97.7% protein, 0.3% fat, 1.8% ash), comprised mainly of β-lactoglobulin and R-lactalbumin, kindly provided by Davisco Foods International (Le Sueur, MN). The locust bean gum (LBG) sample was kindly provided by INDAL (Portugal) and was purified by precipitation with ethanol according to the procedure employed by Lopes da Silva and Gonç alves.34 2.2. Enzymatic Depolymerization of the Galactomannans. An endo-β-mannanase from Aspergillus niger (Megazyme International Ireland, Wicklow, Ireland) was used to cut the β-1,4 linkages within the LBG backbone. The enzymatic hydrolysis was run at 40 °C, under moderated stirring (pH 5, 0.04 U of enzyme/g of galactomannan). Aliquots were taken at selected reaction times and immediately heated at 100 °C for 10 min to denature the enzyme. After cooling, each sample was precipitated in ethanol, washed with ethanol and ketone, dried overnight at 30 °C, and ground to a fine powder. 2.3. Preparation of Solutions. Protein solutions were prepared by adding the WPI powder to Milli-Q Ultrapure water (Millipore Corp., Bedford, MA) and stirring gently at room temperature overnight. Sodium azide 0.02% (w/w) was used in order to avoid bacterial growth. WPI dispersions were centrifuged (10 000g, 15 min, 20 °C) and the pH adjusted to 7.0 using 0.1 M NaOH. The solution was then filtered through a glass sintered filter (G1). The protein concentration was determined from the optical density at 278 nm after suitable dilutions (corrected for turbidity) using the value of 9.6 for the specific absorption coefficient.35 Each LBG fraction was solubilized by stirring at room temperature for 1 h and heating at 90 °C for 30 min, and then centrifuged at 24 400g (30 min, 20 °C). The LBG concentration was determined from the dispersion’s dry matter content (105 °C, overnight). To prepare mixed solutions, the required weights of the LBG and WPI dispersions were mixed at room temperature, under gentle stirring. The WPI was studied. at two concentrations, one close to the sol-gel transition threshold (11% w/w protein) and the other corresponding to a more elastic and well-developed gel (14% protein). The galactomannan content was changed from 0 to 0.7% (w/w). All solutions were degassed under vacuum during 1 h before testing. 2.4. Gel Permeation Chromatography (GPC). GPC was performed to determine the relative molecular weight of the galactomannan samples, using a PL-GPC110 System (Poly-

Monteiro et al.

mer Laboratories Ltd, U.K.) equipped with a differential refractive index detector, two columns in series (PlaguagelOH40 15 µm, 300 × 7.5 mm and Plaguagel-OH60 15 µm, 300 × 7 mm) and a guard column (Plaguagel-OH 15 µm) supplied by Polymer Laboratories Ltd., U.K. The injector and columns were maintained at 40 °C to decrease solvent viscosity and peak broadening. The mobile phase was HPLCgrade water (J. T. Baker Chemical Co.) containing 0.1 M NaNO3 and 0.02% NaN3, with a flow rate of 0.9 mL/min. Native LBG and depolymerized fractions were solubilized as previously described at concentrations in the range of 0.05-0.1% and filtered through a 0.45 µm filter prior to analysis. The injected volume was 100 µL. The analytical columns were calibrated with pullulan standards (Polymer Laboratories, U.K.) in the range of 5.8-1600 kDa. 2.5. Intrinsic Viscosity Measurement. Dilute LBG solutions were prepared over a desired range of concentrations, to have relative viscosities in the range of 1.2-2.0, and filtered through a 0.45 µm syringe filter before measurements were taken. The relative viscosities were determined using a capillary Cannon-Fenske viscometer, at 25.0 ( 0.1 °C. The intrinsic viscosity, [η], was determined by extrapolating to infinite dilution, using the combined Huggin’s and Kraemer’s equations.36 2.6. Measurements of the Mannose-Galactose Ratio. The galactomannan’s composition in neutral sugars was determined by gas-liquid chromatography after sulfuric acid hydrolysis and conversion to their alditol acetates,37,38 in a Carlo Erba 6000 gas chromatograph equipped with a split injector (split ratio 1:60) and a flame ionization detector (FID) detector. The column was a DB-225 (30 m × 0.25 mm, film thickness of 0.25 µm, J & W), and the oven temperature program was 5 min at 220 °C, rising to 230 °C at 20 °C/min, plus 6 min at this temperature. The flow rate of the carrier gas (H2) was set at 1 mL/min at 220 °C. The injector temperature was 220 °C and the FID temperature was 230 °C. 2.7. Rheological Measurements. The rheological measurements were done under small-deformation amplitude using a controlled-stress rheometer (AR-1000, TA Instruments, New Castle, DE) fitted with a cone-plate geometry (diameter of 40 mm and angle of 4°) for isothermal measurements or with a roughish plate-plate geometry (diameter of 40 mm, gap of 1 mm) for nonisothermal measurements. The whey protein solutions and mixtures were submitted to a temperature ramp from 40 to 90 °C at 1 °C/ min, and maintained at 90 °C for 30 min. The temperature was decreased back to 20 °C (rate 1 °C/min), and the viscoelastic properties of the final gel at 20 °C were assessed by frequency sweep experiments, after a short equilibration period (30 min) at this temperature. Isothermal curing experiments were carried out by first performing a 3 h time sweep test at 80 °C, followed by a frequency sweep test at the same temperature. Time and temperature sweep tests were performed at an angular frequency of 5 rad/s and at low strain amplitude of 1%, within the linear viscoelastic regime. The exposed sample surface was covered with a thin layer of mineral oil to avoid evaporation during the measurements. All data reported are the means of at least two (and

Whey Protein-Galactomannan Gelation

Figure 1. Weight-average molecular weight (Mw) and mannose-togalactose ratio as a function of hydrolysis time for LBG samples degraded with β-mannanase (0.04 U of enzyme/g of galactomannan, pH 5, 40 °C).

generally more) replicate tests (two or more different samples). The coefficient of variation was below 8%. 2.8. Confocal Laser Scanning Microscopy (CLSM). The samples for CLSM were noncovalently labeled with fluorescent rhodamine-B (∼ 10 µg/g protein) and their preparation was analogous to the one carried out for rheology. After degassing, the samples were submitted to the same temperature sweep treatment as for the rheological analysis. Images were collected on a Leica SP2 AOBS microscope using a 543 nm He/Ne laser line. The fluorescence signal was collected with a spectral gate of 530-630 nm and processed with ImageJ. 3. Results and Discussion 3.1. Depolymerization of the Galactomannans. Figure 1 shows the changes in weight-average molecular weight (Mw) and in degree of branching (mannose-to-galactose ratio, M/G) as a function of enzymatic hydrolysis time. A significant reduction in molecular weight was observed at short reaction times, followed by a slower decrease at extended hydrolysis. Similar depolymerization patterns were observed for the enzymatic degradation of guar, under similar enzymatic hydrolysis conditions.39 During the course of the galactomannan depolymerization, the M/G of the degraded samples remained almost unchanged, indicating that the (1f6) linkages of the galactose side chains were not significantly affected. The original purified LBG sample, treated under the same conditions as the depolymerized samples but in the absence of enzyme, and the samples obtained for 10, 120, and 960 min of enzymatic hydrolysis, were selected to study the galactomannan’s molecular weight effect on WP gelation. Figure 2 shows the molecular weight distribution patterns for the selected LBG samples. The relative weight-average (Mw) and number-average (Mn) molecular weights were determined from the GPC elution curve. The estimated molecular characteristics (Mw, Mn, [η] and M/G) of the selected samples are summarized in Table 1. As expected, the intrinsic viscosity ([η]) of the LBG fractions decreased as a consequence of the decreasing molecular weight. Contrary to what was previously observed for the enzymatic

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Figure 2. Molecular weight distribution patterns obtained for LBG samples depolymerized to different extents with β-mannanase (0.04 U of enzyme/g of galactomannan, pH 5, 40 °C). Results correspond to different hydrolysis times: A, 0 h; B, 10 min; C, 2 h; D, 16 h.

depolymerization of guar,39,40 the molecular weight distribution did not change considerably with the degradation time, suggesting that in the case of LBG a more random enzymatic degradation of chains may occur. This can be attributed to a high accessibility of the enzyme to the mannan chain, due to the low degree of branching of the LBG and/or to the different distribution pattern of the galactose residues occurring along the mannan backbone of the LBG and guar polysaccharides.41 3.2. The effect of LBG on Incipient WP Gels. 3.2.1. Microstructure. The microstructure of WP gels was studied using confocal laser scanning microscopy. Clear evidence for phase separation occurring at a micrometer level was found for the mixtures with the LBG fractions, except for that with the lowest molecular weight. The CLSM pictures in Figure 3 show how variations in the molecular mass and/ or content of LBG influenced the incipient network structure of the WP. White and light gray areas in the pictures represent protein phase while black areas represent the polysaccharide-rich phase. Fluorescence was regularly distributed in the micrograph of WP alone, indicating that the protein molecules were homogeneously distributed in the sample. Clearly, the phase mass ratio and the LBG molecular weight influence the microstructure. The galactomannan with the lowest molecular mass (LBG D) did not change the microstructural arrangement of the protein network at this length scale. However, for the mixtures with galactomannan samples with higher molecular weights (LBG A-C), phase separation occurred with irregular masses of polysaccharide solution dispersed in a continuous protein matrix. Similar emulsion-like microstructures have been observed for other protein-polysaccharide mixtures.42-46 The size of the polysaccharide-rich areas and the degree of connection between them increase with increasing galactomannan concentration or molecular weight, probably associated to an increase in the volume of the dispersed phase. Interconnection between the polysaccharide-rich droplets occurred for the higher LBG molecular weight and concentration analyzed, originating bicontinuous phaseseparated systems.

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Table 1. Main Molecular Characteristics of the Selected LBG Samples Degraded to Different Extents with β-Mannanasea

a

sample

degradation time (min)

[η] (dL/g)

Mw (Da × 10-5)

Mn (Da × 10-5)

M/G

LBG A LBG B LBG C LBG D

0 10 120 960

13.8 ( 0.3 7.0 ( 0.2 5.8 ( 0.1 2.6 ( 0.1

19.3 7.86 4.25 1.68

17.3 7.67 3.93 1.59

3.84 ( 0.02 3.85 ( 0.02 3.82 ( 0.02 3.81 ( 0.03

Mean ( standard deviation for triplicate measurements; 0.04 U of enzyme/g of galactomannan, pH 5, 40 °C.

Figure 4. G′ (filled symbols) and G′′ (open symbols) as a function of isothermal curing time at 80 °C (ω ) 5 rad/s) for 11% WPI alone and WPI/LBG blends with 0.7% LBG (fractions A and D).

Figure 3. Confocal micrographs of final gel states for 11% WPI gels containing 0.25% and 0.70% galactomannan with different molecular masses, LBG samples A, B, C, and D. Each image width is 175 µm.

3.2.2. Viscoelasticity. WP alone and in mixture with the LBG fractions at different concentrations was subjected to isothermal curing at 80 °C for 3 h. Representative curves for the time dependence of the viscoelastic moduli, storage (G′) and loss (G′′) moduli, obtained for 11% WP/0.7% LBG mixtures, fractions A and D, are shown in Figure 4. The mixtures with the other LBG molecular weight fractions investigated lay in between. The same experiments were performed for mixtures with different LBG concentrations.

The WP dispersion at 11% did not gel under the thermal and kinetic history analyzed. Despite the scatter of the experimental moduli values, due to the closeness to the lower limit of displacement detection of the rheometer, it is interesting to note that G′ is already higher than G′′ at the beginning of the measurement, but then it decreases slightly with time, whereas G′′ increases, with the crossover occurring around 2 h of curing. Similar peculiar behavior was previously observed for globular proteins in diluted solution after heat treatment7,47 and can be attributed to the presence of protein aggregates. In the presence of the galactomannan, and for the lower concentrations analyzed (0.25 and 0.5%), both viscoelastic moduli did not change significantly during curing, with G′′ always higher than G′, reflecting essentially the viscoelastic behavior of the polysaccharide. Significant changes were observed only for the higher molecular weight LBG sample at 0.5% and for all fractions at 0.7%. Both G′ and G′′ increase more rapidly at first, more pronounced as the LBG molecular weight increases, and then slope down increasing slightly with time (Figure 4). The G′-G′′ crossover occurred around the first 5 min for the mixture with LBG A, whereas for the lowest molecular weight (LBG D) the crossover seems to occur close to the end of the 3 h curing period. These data show that close but below the gelling conditions of the WP, the presence of the nongelling polysaccharide at the highest concentration analyzed induces gelation to occur. To study the influence of temperature on the rheological properties of the WP/galactomannan systems, continuous temperature sweeps were carried out (1 °C/min). For 11% WP alone, the viscoelastic moduli values during heating to 90 °C, and cooling back to 20 °C, were close to the rheometer resolution. At 20 °C, G′′ was still higher than G′, as illustrated in Figures 6 and 7, but G′ already tends to level



Figure 7. Mechanical spectra of 11% WPI/LBG blends at 20 °C, showing the variation of G′ (9) and G′′ (0) with oscillatory angular frequency (ω). Results for the LBG sample with the highest molecular weight, LBG A, at different concentrations (%, w/w). Numbers in the figure indicate polysaccharide concentration. Figure 5. Changes in G′ (filled symbols) and G′′ (open symbols) during temperature sweep experiments (ω ) 5 rad/s, 1 °C/min) for 11% WPI + LBG mixtures. Increasing molecular weight of the LBG sample (0.7%, w/w) from A to D: (A) LBG D, (B) LBG C, (C) LBG B, (D) LBG A.

Figure 6. Mechanical spectra of 11% WPI alone and WPI/LBG blends at 20 °C, showing the variation of G′ (9) and G′′ (0) with oscillatory angular frequency (ω). Increasing molecular weight of the LBG sample (0.7%, w/w) from A to D: (A) LBG D, (B) LBG C, (C) LBG B, (D) LBG A. Small symbols and lines denote the behavior of WPI alone.

off toward the low-frequency range and to cross the G′′(ω) curve, suggesting a solidlike behavior, despite the very low moduli values. In this case, the WP system behaves as a structured viscoelastic fluid, probably close to the critical gel point, due to limited macromolecular aggregation. Similar heating profiles were observed in the presence of the lower galactomannan concentrations and molecular weights, but in this case, during cooling, G′ was higher than G′′. The influence of the galactomannan molecular weight on the heating-cooling viscoelastic profiles is illustrated in Figure 5 for 11% WP, in the presence of 0.7% LBG fractions.

The viscoelastic moduli initially decrease, but then a sharp increase is observed for a temperature around 70 °C, more pronounced as the galactomannan molecular weight increases. Cooling the gels to 20 °C causes both moduli to increase around a factor of 5, without any appreciable change on their viscoelastic character. At the higher galactomannan molecular weight, a significant difference on the moduli values is already observed before the stepped increase, with G′′ > G′ and both showing higher values. Also, in this case, during cooling the difference between G′ and G′′ is smaller, meaning that the system has now a more pronounced viscous character. Figure 6 shows the viscoelastic profile of the gels after cooling back to 20 °C. In the presence of the LBG fractions the viscoelastic behavior of the gels changed dramatically. Increasing the molecular weight from 168 to 786 kDa resulted in higher moduli with G′ less dependent on frequency (Figure 6A-C). However, for the higher molecular weight analyzed (Figure 6D), G′ decreased slightly, its dependence on frequency increased (slope ∼ 0.23, against 0.12 in Figure 6C), and a smaller separation between G′ and G′′ was observed, i.e., an elastically less perfect network was formed. One may presume that there will be a molecular weight critical limit, depending on concentration, until the gel is strengthened without loosing/decreasing its elastic character. This optimum limit may be related to the formation of a bicontinuous network with lower connectivity within the protein-rich gelled phase. Although at the micrometer level no appreciable influence of the low molecular weight LBG fraction was observed (Figure 3), its presence clearly influenced the viscoelastic behavior of the mixed system. The systems with the low molecular weight LBG fractions D and C (Figure 6 A-B), despite their very different microstructures (Figure 3), show quite similar viscoelastic profiles, characteristic of tenuous gelled networks, with G′ leveling off toward the lowfrequency range but increasing toward the highest frequencies, i.e., an elastic plateau at long observation times but a tendency to flow at higher frequencies. For the mixed gels with LBG fractions B and A (Figure 6 C-D), those that exhibited the bicontinuous phase-

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Figure 9. Changes in G′ with time, showing the effect of LBG addition on WPI isothermal (80 °C) gel formation. LBG samples with three different molecular weights (LBG A (2, 4), LBG C (b, O), LBG D ([, ])) at two different concentrations, 0.25% (open symbols) and 0.7% (filled symbols); (9) denotes the WPI alone.

Figure 8. Confocal micrographs of final gel states for 14% WPI gels containing 0.25 and 0.70% galactomannan with different molecular masses, LBG samples A, B, C, and D. Each image width is 175 µm.

separated microstructures (Figure 3), the elastic plateau at low frequencies disappeared, and the G′ traces are essentially straight. The effect of the polysaccharide concentration, LBG fraction A, on the frequency dependencies of the viscoelastic moduli obtained at 20 °C is illustrated in Figure 7. It is clearly shown that structuration of the system increases as the LBG concentration increases: both moduli increase, G′ levels off at low frequencies, crossing G′′, and then the spectra exhibit much weaker frequency dependence. 3.3. The Effect of LBG on 14% WP Gels. 3.3.1. Microstructure. Figure 8 shows the CLSM pictures obtained for the WP alone at 14% and WP/LBG mixtures, for two different LBG concentrations and four different molecular weights. The galactomannan with the lowest molecular weight (LBG D) has also not changed the microstructural arrangement of the protein network. For the mixtures with

higher molecular weight LBG fractions, the microstructural analysis revealed the expected phase-separated systems, one containing mainly whey protein, whereas the other one was enriched with gums. At 0.25% LBG, a higher polysaccharide molecular weight led to phase separation and a WPI gel (light domain) filled with dispersed droplets (dark domains) of nongelled polysaccharide. As the polysaccharide concentration and/or molecular weight increase the phase-separated systems evolve from one comprising unconnected polysaccharide-rich droplets suspended in a continuous protein-rich gelled matrix to a more interconnected morphology. At the higher concentration and molecular weight, the dispersed droplets connect through fusion into an interpenetrating phase, forming a bicontinuous system. Comparison with the microstructures observed for 11% WPI (Figure 3), at the same polysaccharide concentration and molecular weight, it can be concluded that the microstructural organization depends also on the protein network. For the mixtures with the LBG C sample, at 14% WPI the polysaccharide-rich droplets seem to be isolated, dispersed in the protein matrix, while at 11% WPI they were already more connected. For the 11% WPI systems, the dispersed polysaccharide inclusions are more irregular, and it seems that more extensive connectivity occurred between them, i.e., increasing the protein network density seems to delay the growth and connectivity between the polysaccharide dispersed phase. 3.3.2. Viscoelasticity. In Figure 9, the storage modulus (G′) is plotted as a function of curing time at 80 °C for the 14% WP alone and WP/LBG mixtures at pH 7.0. The development of the storage modulus (G′) with time follows the general behavior observed in many biopolymers’ gelation processes including previously reported data for whey protein gelation.3,48,49 During the first curing step at 80 °C, G′(t) increased rapidly in the first 30 min, after which the evolution was slower and tended to an asymptotic value. However, after 3 h of curing the gelled systems are still evolving and not at equilibrium; hence, the mechanical spectra recorded at this temperature after 3 h of curing (Figure 10) have to be considered under caution. Despite the similar curing profile, the influence of the LBG addition is clearly evident. A faster increase of G′ is observed at the start of the curing,


Figure 10. Mechanical spectra of 14% WPI/LBG blends at 80 °C, showing the variation of G′ (filled symbols) and G′′ (open symbols) with oscillatory angular frequency (ω). Results for 0.25% (A) and 0.7% (B) LBG fractions, with different molecular weights: (2, 4) LBG D; (9, 0) LBG C; ([, ]) LBG A. Also shown is the behavior of 14% WPI alone (b, O).

and G′ reaches higher values as the polysaccharide concentration and/or molecular weight increase. Figure 10 shows the mechanical spectra measured after curing the gels at 80 °C. At 0.25% added galactomannan fractions, the general viscoelastic profile is retained, with a general increase of both moduli as the molecular weight increases, but similar frequency dependences, and with a minimum in G′′(ω) located at a similar frequency value (∼0.6 rad/s). At 0.7% galactomannan, a more pronounced increase in G′ is observed for all fractions. For the higher molecular weight (LBG A), a change on the viscoelastic profile is noted, with a slightly higher dependence of G′ on frequency and the appearance of a loss peak, meaning that more appreciable relaxation processes are occurring within the scanned time scale, i.e., more molecular rearrangements are now allowed. The nonisothermal gel formation of the WP/LBG samples during controlled heating (1 °C/min) from 40 to 90 °C, holding at this temperature for 30 min, and then cooling back to 20 °C (results not shown), followed the general profile previously observed.32 A rapid increase in G′ is observed after a critical temperature is reached; G′ also increases strongly during the holding time at 90 °C, and then a smoother increase occurred during the cooling step. Addition of the galactomannan samples did not change the general profile of the G′ dependence on temperature. However, generally higher G′ values and lower gelation temperatures were observed than for WP alone. Changes in the viscoelastic behavior of the WP/LBG mixtures close to the gelation point, defined by the characteristic parameters of the sol-gel transformation, G′ and G′′ moduli, are exemplified in Figure 11. Even considering that these systems are not at equilibrium and that the kinetics would influence the observed behavior, the presence of the galactomannan and its molecular weight clearly influence the gelation onset of the WP dispersion. Considering the first increase in G′ as the criterion to define the gelation temperature,50 it is observed that this critical temperature decreases as the galactomannan molecular weight increases. This means that increasing the polysaccharide’s concentration and/or molecular weight somehow


Figure 11. Temperature dependence of G′ (filled symbols) and G′′ (open symbols), for 14% WPI + LBG mixtures at pH 7, heated from 40 °C to 90 °C (ω ) 5 rad/s, 1 °C/min): (9, 0) WPI alone; ([, ]) 0.7% LBG D; (2, 4) 0.7% LBG A.

promote sufficient protein aggregation to form a gel at lower temperature. At the higher galactomannan molecular weight and concentration, a significant difference on the moduli values is already observed before the stepped increase, with G′′ > G′ and both showing higher values, mainly determined by the increase in loss processes due to the inclusion of the high molecular weight nongelling polymer. Figure 12 shows the mechanical spectra measured after cooling the gels back to 20 °C, for 0.25% and 0.7% LBG concentrations and different molecular weights. The behavior of other LBG concentrations lay in between. Generally, these mechanical spectra are typical of reasonably strong gels with pronounced elastic character, with G′ > G′′ by about one decade, so tan δ (G′′/G′) is close to 0.1, both moduli are almost parallel, and G′ shows a slightly positive slope around 0.06-0.07. At 0.25% LBG fractions, the main observed effect on the viscoelastic profile of the WP gel alone was the slight increase in G′ and decrease of tan δ, i.e., a slight reenforcement of the gel. The mechanical properties scanned at small dynamic deformations seem quite insensitive to the microstructural particularities observed for these systems (Figure 8). At 0.7% LBG fractions, a more pronounced effect of the polysaccharide molecular weight was observed. The G′ for the mixtures is always higher than for the WP alone, but a progressive increase in molecular weight of the added LBG decreases the elastic character of the system. For the mixed gel with LBG fraction B, the gel is still more elastic than the WP alone at the low frequencies but now with a more viscous character than the protein gel over short time scales. For the mixed gel with LBG fraction A, the highest molecular weight, a more dissipative network was formed, with a slope of G′ versus frequency of around 0.11, higher tan δ, and a loss peak around 1 rad/s, in close agreement with that observed for the same system under isothermal curing at 80 °C (Figure 10). The additional relaxation processes observed within the observation time of the frequency scan are probably related to the less-connected protein network observed by CLSM. 3.4. Microstructure versus Rheology. The smallamplitude mechanical spectra seem to be insensitive to the

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strength of the protein chain bundles forming the elastic active junctions. Connectivity seems to play an important role, as emphasized previously for amylopectin/β-lactoglobulin particulate mixed gels.43 4. Conclusions

Figure 12. Mechanical spectra of 14% WPI alone and WPI/LBG blends at 20 °C, showing the variation of G′ (9), G′′ (0) and tan δ (×) with oscillatory angular frequency (ω). Increasing molecular weight of the LBG samples (0.25% and 0.7%) downward. Lines denote for the behavior of WPI alone.

changes occurring in the microstructure, as the system evolves from homogeneous at the micrometer scale to emulsion-like phase separated, and then when the dispersed polysaccharide-rich droplets start to interconnect, especially at the higher WP concentration analyzed, more structured protein networks. This lack of correlation between microstructure and small-deformation rheology was also highlighted in other works.24,43,51 Considering the similarities of the viscoelastic profile shown in Figure 6 (A and B), and the different microstructures displayed by the same systems (micrographs in Figure 3, for the mixtures with 0.7% LBG D and C), or the similar mechanical spectra shown in Figure 12 with the very different microstructures displayed in Figure 8, e.g., for the mixtures with 0.7% LBG fractions D, C, and B, we may conclude that the linear viscoelasticity seems to be insensitive to some of the microstructural changes observed within the mixed gels. Rheology is likely to be dependent on a complex balance between connectivity of the network and density/

The experimental results presented here outline the important effect of a neutral polysaccharide on the heatinduced gelation of WP, focusing on concentration and molecular weight effects of the nongelling polymer. This study clearly showed that molecular weight is a limiting factor for phase separation to occur and that the resulting microstructures will depend on both galactomannan molecular weight and protein-to-polysaccharide ratio. One can presume that the two WP fine-stranded networks we have studied, at 11% and 14% protein concentration, are characterized by similar macromolecular organization, formed by strands of relatively long protein chains as previously described2 and that the main dissimilarities are the limited aggregation and less-extended cross-linking in the first case, not enough to form a gel, whereas a dense gelled network is formed in the latter case. The presence of the nongelling polysaccharide during the heat-induced gelation of the protein has a clear effect on the protein aggregation and gelation, which we assign mainly to thermodynamic incompatibility between the two polymers. As this mechanism implies volume exclusion between these two macromolecules, the influence of increasing molecular weight of the nongelling polymer is thus attributed to enhanced volume exclusion phenomena. Further, steric exclusion at high levels of polysaccharide’s hydrodynamic volume results in biphasic networks. The increase of the effective concentration of both polymers will originate a more dense fine-stranded structure (increasing the coarseness of the elastic active strands), explaining the observed induced gelation at the lower WP concentration and the general increase in the storage modulus/stiffness for the gelled mixtures. The dispersed polysaccharide-rich phase act as active filler that reinforces the protein network and increases the modulus, as far as a critical connectivity is maintained. At the higher molecular weight and concentration of the nongelling polymer, the protein network starts to lose elastic perfection, the viscoelastic profile shows more marked loss processes, probably related to the formation of a bicontinuous structure with lower connectivity. We trust that these findings may suggest strategies for controlling the rheological behavior of WPbased food products and for manipulating microstructures for new or improved applications. Acknowledgment. We thank Fundaç aõ para a Cieˆncia e Tecnologia (Lisboa, Portugal) for financial support through the programs POCTI and FEDER (Project POCTI/33626/ BIO/2000). We also thank INDAL (Faro, Portugal) and Davisco Foods International, Inc. (Le Seur, MN) for providing the LBG and WPI samples, respectively. References and Notes (1) Foegeding, E. A.; Davis, J. P.; Doucet, D.; McGuffey, M. K. Advances in modifying and understanding whey protein functionality. Trends Food Sci. Technol. 2002, 13, 151-159.

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Influence of Galactomannans with Different Molecular Weights on

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